The present invention relates to the electrophoresis separation of mixtures of molecular particles, such as nucleic acids or proteins and pertains particularly to an improved bufferless method and apparatus for the electrophoresis separation of nucleic acids, proteins and the like.
A great deal of laboratory research is carried out wherein separation of DNA, RNA or protein is widely utilized. This process is widely used in research laboratories for analysis or separation of restriction digest fragments of DNA, where polvmerase chain reaction DNA products are analyzed or prepared, where synthetic oligo nucleotides are analyzed or prepared, where protein are analyzed and/or blotted, or any application involving small scale separation of nucleic acid. Electrophoresis separation is typically carried out in a separation medium, such as a gel of agarose or acrylamide. These gels are typically cast in molds consisting of two glass plates to form a slab or in glass tubes to form a cylinder.
Molecular weight or size separation is typically achieved by means of a gel composition formed to create a network of pour sizes which provides a seiving action to separate the molecules. In order to effect the electrophoretic separation, means for connecting an electric field to the separating gel is needed. This is usually accomplished by immersing the ends of the gel slab or cylinder in reservoirs of electrically conductive buffer. These buffers are connected by platinum or carbon electrodes immersed in the fluid to the positive and negative terminals of a power supply, which establishes a voltage gradient of from twenty to three-thousand volts across the separating gel to draw the molecules in the mixture through the gel matrix. This method of attaching the gel to the power supply requires large volumes of buffer to fill reservoirs, immersion of the gel in the buffer or connection via wicks and bulky apparatus for electrophoresis.
One of the major drawbacks of electrophoresis has been the use of these buffers and the complicated equipment required for them. The buffers must be carefully prepared and handled. The buffers are also subject to evaporation with resultant loss of effectiveness due to Ph changes.
Various attempts have been made in the past to overcome the problems inherent in the use of liquid buffer solutions. One such approach is shown for example in U.S. Pat. No. 3,715,295, issued Feb. 6, 1973 to Tocci. This patent describes a device in which wells are formed on each end of the gel, and a semi-solid buffer is placed in the wells over electrodes disposed in the wells. This arrangement, however, still requires large and complicated equipment.
Another attempt to solve this problem is disclosed in U.S. Pat. No. 3,865,712, issued Feb. 11, 1975, which discloses the use of a filter paper as a wick saturated with a buffer placed over the gel. Electrodes are positioned on the filter paper, with electrodes pressed into contact with the filter paper. One problem with this approach is that the wick materials become dried out, with a resultant loss of electrical continuity.
Other attempts to eliminate the use of buffers have included apparatus having its electrodes embedded within the gel. Such known apparatus may be operated at very low voltages, and the current densities must also be kept very low, with the result that the reaction time takes considerable time. Otherwise, the gel surrounding the electrodes would melt and consequently the supply of current would be interrupted since heat cannot be abducted.
U.S. Pat. No. 4,443,319, granted Apr. 17, 1984 to Chait et al discloses a thin film electropheretic device formed with layers with a substrate, electrodes on the substrate and a gel form over both. This approach is suggested for use of very thin open face films oriented in a horizontal plane with very thin films and very low voltage
It is therefore desirable that an improved electrophoresis device be available which eliminates the need for buffers and can also be operated at sufficiently high voltages to obtain rapid results.